1. Volume 24, Issue 10, Pages 1679-1692 (October 2016)
Tankyrase-1 Ankyrin Repeats Form an Adaptable Binding Platform for Targets of ADP- Ribose Modification 
Travis Eisemann, Michael McCauley, Marie-France Langelier, Kushol Gupta, Swati Roy, Gregory D. Van Duyne, John M. Pascal 
Structure 
Volume 24, Issue 10, Pages (October 2016)
DOI: /j.str
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2. Structure 2016 24, 1679-1692DOI: (10.1016/j.str.2016.07.014)
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3. Figure 1 Crystal Structure and Solution Parameters of TNKS ARC1–3
(A) Schematic of TNKS domain boundaries and construct map.
(B) Normalized P(r) distribution plots for ARC1–3, ARC4–5, and ARC1–5. Error propagated from Monte-Carlo simulations as implemented in GNOM.
(C) Crystal structure of ARC1–3 bound to IRAP peptide (chain A).
(D) Ab initio bead model of ARC1–3 in complex with IRAP (gray) overlaid with crystal structure.
(E and F) Inter-ARC linker structures (yellow) showing the broken helix structure of the ARC1 (red) to ARC2 (green) linker (E) and the continuous helix structure of the ARC2 to ARC3 (blue) linker (F). Residues supporting these interfaces are labeled.
See also Figures S1–S3 and Table S1.
Structure  , DOI: ( /j.str )
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4. Figure 2 Binding Capacities of Individual ARCs
(A and B) Binding pocket and IRAP peptide interactions (red dotted lines) of ARC1 (A) and ARC2 (B).
(C) Disrupted pseudo binding pocket of ARC3, with the outline of modeled bound peptide (black trace) highlighting steric clashes that prevent binding.
(D) ARC1–5 mutagenesis target residues representing the quadruple mutant xx3xx.
(E) TNKS binding motifs of Axin1 (top) and IRAP (bottom). Binding motifs are underlined, and the Arg and Gly residues at the N and C termini are shown in bold.
(F) Schematic of Axin1-derived peptide library.
(G–I) FP binding analysis of individual ARC interaction with IRAP (G), Axin-SEG1 (H), and Axin-SEG2 (I). Data represent mean ± SEM.
See also Figure S4 and Table S2.
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5. Figure 3 Bivalent Binding to ARCs 1/2 and 4/5
(A and D) FP binding analysis of Axin-WT binding to ARC pair 1/2 (A) and 4/5 (D).
(B and E) FP binding analysis of Axin-NL binding to ARC pair 1/2 (B) and 4/5 (E). Gray lines represent either or xx3xx constructs, dashed black lines represent constructs with single functional ARCs, and colored solid lines represent constructs with functional ARC pairs.
(C) Block diagram representing Axin-WT bivalent binding mode with ARC1/2. The N terminus (N) and C terminus (C) of the peptides are labeled to provide the orientation of the ARC binding sites. Based on our binding affinity measurements, Axin-SEG1 is shown bound to ARC2, and Axin-SEG2 is bound to ARC1, although the reverse binding mode is theoretically possible.
(F) Block diagram representing Axin-WT bivalent binding mode with ARC4/5. Axin-SEG1 is shown bound to ARC4, and Axin-SEG2 is bound to ARC5, based on our binding affinity measurements; however, a reversed binding mode is theoretically possible.
Data represent mean ± SEM. See also Table S2.
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6. Figure 4 Bivalent Binding to ARCs 2/4 and 2/5
(A and C) FP binding analysis of Axin-WT binding to ARC pair 2/4 (A) and 2/5 (C).
(B and D) FP binding analysis of Axin-NL binding to ARC pair 2/4 (B) and 2/5 (D). Gray lines represent either or xx3xx constructs, dashed black lines represent constructs with single functional ARCs, and colored lines represent constructs with functional ARC pairs.
(E) Block diagram representing Axin1 bivalent binding to ARC2/5. Due to its high relative affinity, Axin-SEG1 is shown bound to ARC2 while Axin-SEG2 is bound to ARC5. The linkers for Axin-WT (gray) and Axin-NL (yellow) are shown between Axin1 binding segments.
(F) Binding data for fluorescein isothiocyanate (FITC)-Axin-WT and FITC-Axin-NL versus ARC1–5 constructs.
Data represent mean ± SEM. See also Table S2.
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7. Figure 5 Analytical Ultracentrifugation Analysis of ARC1–5
(A–D) Sedimentation velocity analysis of ARC1–5 apo (A) or in complex with Axin-SEG1 (B), Axin-WT (C), or Axin-NL (D). The top panels show absorbance data (circles) and associated c(S) model fit (lines). The bottom panels show residuals for the fit.
(E) c(S) distributions derived from sedimentation velocity experiments.
(F) Parameters derived from sedimentation velocity and equilibrium experiments.
See also Figure S4.
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8. Figure 6
ARC1–5 Solution Scattering and Modeling of Solution Conformations
(A) ARC1–5/peptide scattering profiles with close-up of q range (inset). Error bars represent plus and minus the combined standard uncertainty of the data collection.
(B) Normalized P(r) distribution plots for ARC1–5/peptide samples. Error propagated from Monte-Carlo simulations as implemented in GNOM.
(C) χ2 versus Rg plot for ARC1–5 in complex with Axin-WT (green), Axin-SEG1+2 (red), and Axin-NL (blue) from the SASSIE model library. For each dataset, the 20 models with the lowest χ2 values are marked with solid circles.
(D) Ensemble conformations for ARC1–5 in complex with Axin-WT (green). A poor-fitting model (Rg = 52.3 Å, χ2 = 19.0) is shown in gray outline.
(E) Ensemble conformations for ARC1–5 in complex with Axin-SEG1+2 (red). Axin-WT ensemble conformations are represented by a green outline. A poor-fitting model (Rg = 54.2 Å, χ2 = 10.8) is shown in gray outline.
(F) Ensemble conformations for ARC1–5 in complex with Axin-NL (blue). Axin-WT (green) and Axin-SEG1+2 (red) conformations are represented by outlines. A poor-fitting model (Rg = 49.4 Å, χ2 = 24.1) is shown in gray outline.
All ensemble conformations in (D–F) represent the 20 best fits from (C), and were aligned to ARC1–3 (black). Rg values represent an average of the top 20 models from (C), using Rg as calculated from SASSIE. ARC2 to ARC5 distance measurements represent an average of the distance from Y412 at the end of the ARC2 peptide binding pocket to W902 at the start of the ARC5 peptide binding pocket. See also Figures S5–S7.
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9. Figure 7 Model for ARC1–5:Axin1 Binding Mode
(A) Representation of the inherent (top panel, back view) and substrate-dependent (bottom panel, side view) conformational sampling of the TNKS ankyrin repeat domain.
(B) Model of Axin1-TNKS binding mode. Alternative substrate positioning resulting from ARC1/2 (orange) or ARC2/5 (blue) binding is shown.
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